Agar is a linear polysaccharide obtained from the cell wall of some red algae and is composed of agarose and agaropectin. Agarose consists of disaccharide units of 3-O-linked β-D-galactopyranose and 4-O-linked 3,6-anhydro-α-L-galactose. Agarose can be hydrolyzed by agarases. Agarases can be classified into two groups, i.e., α-agarases and β-agarases, according to their enzymatic cleavage sites, as α-agarases cleave the α-1,3 linkage in agarose and β-agarases cleave the β-1,4 linkage in agarose. Agar can be hydrolyzed by acids or α-agarases to obtained agaro-oligosaccharides having a 3,6-anhydro-α-L-galactose group at the reducing end. On the other hand, β-agarases can hydrolyze agar into neoagaro-oligosaccharides having a D-galactose group at the reducing end. Neoagaro-oligosaccharides can only be produced by enzymatic catalysis, not by a chemical method.
β-Agarases can be classified into three glycoside hydrolase families, GH16, GH50 and GH86, on the basis of their amino acid sequences. Most β-agarases belong to family GH16, which can hydrolyze agar and neoagarohexaose and the main product is neoagarotetaose [see Schroeder et. al., (2003) Investigation of the role of a β(1-4) agarase produced by Pseudoalteromonas gracilis B9 in eliciting disease symptoms in the red alga Gracilaria gracilis. Microbiology 149: 2919-2929, and Allouch et. al., (2003) The three-dimensional structures of two β-agarases. J. Biol. Chem. 278: 47171-47180]. A representative β-agarase in family GH50 is AgaA protein obtained from Vibrio sp., which can hydrolyze neoagarotetaose and the main product is neoagarobiose [see Sugano et. al., (1993) Cloning and sequencing of agaA, a unique agarase 0107 gene from a marine bacterium, Vibrio sp. strain JT0107. Appl. Environ. Microbiol. 59: 3750-3756]. Representative β-agarases in family GH86 are AgrA protein produced by Pseudoalteromonas atlantica T6c [see Belas et. al., (1988) Cloning and gene replacement mutagenesis of a Pseudomonas atlantica agarase gene. Appl. Environ. Microbiol. 54: 30-37], and AgaO protein obtained from Microbulbifer-like bacterium, strain JAMB-A94. AgaO can hydrolyze agar and the main end product is neoagarohexaose [see Ohta et. al., (2004) Cloning, expression, and characterization of a glycoside hydrolase family 86 β-agarase from a deep-sea Microbulbifer-like isolate. Appl. Microbiol. Biotechnol. 66: 266-275].
Neoagaro-oligosaccharides have been widely used in food, cosmetic and medical industries. It is found that neoagaro-oligosaccharides can reduce starch decomposition rate and inhibit the growth of bacteria, and thus can be used as low-calorie food additive. In addition, the polysaccharides produced by hydrolyzing marine algae with β-agarases can stimulate the activation of macrophages, and thus can be used as a functional food to enhance immune response [see Yoshizawa et. al., (1995) Macrophage stimulation activity of the polysaccharide fraction from a marine alga (Porphyra yezoensis): structure-function relationships and improved solubility. Biosci. Biotechnol. Biochem. 59: 1933-1937]. Furthermore, neoagarobiose produced from β-agarases hydrolysis has skin moisturizing and whitening effects [see Kobayasgu et. al., (1997) Neoagarobiose as a novel moisturizer with whitening effect. Biosci. Biotechnol. Biochem. 61: 162-163]. In molecular biology laboratories, agarases is useful in the purification of DNA by agarose electrophoresis, or in the manufacture of protoplasts of algae [see Araki et. al., (1998) Optimization of parameters for isolation of protoplasts from Gracilaria verrucosa (Rhodophyta). J. Mar. Biotechnol. 6: 193-197].
However, the activity, stability and yield of the conventional β-agarases produced by the current methods are not satisfying. There is still a need for a new β-agarase with better activity, stability and yield.
Thalassomonas agarivorans was isolated by Dr. Shieh Wung Yang's laboratory at National Taiwan University [see Jean et. al., (2006) Thalassomonas agarivorans sp. nov., a marine agarolytic bacterium isolated from shallow coastal water of An-Ping Harbour, Taiwan, and emended description of the genus Thalassomonas. Int. J. Syst. Evol. Microbiol. 56:1245-1250]. It was found that Thalassomonas agarivorans could hydrolyze the agarose in a culture plate, but the type of the agarase produced thereby was unknown. Before Jean et al. (2006), the only strain in the genus Thalassomonas known to have agarolytic activity was Thalassomonas sp. strain JAMB-A33 [see Hatada et. al., (2006) Hyperproduction and application of α-agarase to enzymatic enhancement of antioxidant activity of porphyran. J. Agric. Food Chem. 54: 9895-9900], and the agarase of Thalassomonas sp. strain JAMB-A33 was an α-agarase. Therefore, prior to the invention, no Thalassomonas strains was identified to have the ability to produce β-agarase.